Enhancers such as acetosyringone

ABSTRACT

PCT No. PCT/DK95/00384 Sec. 371 Date Feb. 20, 1997 Sec. 102(e) Date Feb. 20, 1997 PCT Filed Sep. 27, 1995 PCT Pub. No. WO96/10079 PCT Pub. Date Apr. 4, 1996The invention is directed to a method of bleaching dye or colorant in solution which comprises a phenol oxidizing enzyme (e.g. A peroxidase or a laccase) and an enhancing agent (e.g. acetosyringone).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK95/00384 filed Sep. 27, 1995 and claims priority under 35 U.S.C.119 of Danish application 1109/94 filed Sep. 27, 1994, 0952/95 filedAug. 25, 1995 and 1044/95 filed Sep. 19, 1995, the contents of which arefully incorporated herein by reference.

FIELD OF INVENTION

The invention relates to a method of oxidizing a compound with a phenoloxidizing enzyme and an enhancing agent. The invention also relates to adetergent additive and to a detergent composition.

BACKGROUND ART

By a phenol oxidizing enzyme is meant an enzyme which by using hydrogenperoxide or molecular oxygen, is capable of oxidizing organic compoundscontaining phenolic groups. Examples of such enzymes are peroxidases andoxidases.

It has earlier been found that coloured substances leached from dyedfabrics could be bleached by means of a phenol oxidizing enzyme. The useof peroxidases or oxidases for inhibiting dye transfer in this way isdescribed in WO 91/05839.

Certain oxidizable substances, e.g., metal ions and phenolic compoundssuch as 7-hydroxycoumarin, vanillin, and p-hydroxybenzenesulfonate, havebeen described as accelerators or enhancing agents able to enhanceenzymatic bleaching reactions (cf. e.g. WO 92/18683, WO 92/18687, andKato M and Shimizu S, Plant Cell Physiol. 1985 26 (7), pp. 1291-1301(cf. Table 1 in particular)). In WO 94/12621 other types of enhancingagents are disclosed, e.g., phenothiazines and phenoxazines.

It is the object of this invention to provide a new group of enhancingagents which are effective for enhancing phenol oxidizing enzymes.

SUMMARY OF THE INVENTION

It has now surprisingly been found that a new group of organic chemicalsubstances performs excellently as enhancers of phenol oxidizingenzymes.

This new group of organic chemical substances not only make thebleaching reactions faster compared with using the phenol oxidizingenzyme alone, but many compounds which could not be bleached at all, maynow be bleached by using the method of the invention.

Accordingly, the invention provides a method of oxidizing a compoundwith a phenol oxidizing enzyme, characterized by the presence of anenhancing agent of the following formula: ##STR1## in which formula A isa group such as --D, --CH=CH--D, --CH=CH--CH=CH--D, --CH=N--D, --N=N--D,or --N=CH--D, in which D is selected from the group consisting of--CO--E, --SO₂ --E, --N--XY, and --N⁺ --XYZ, in which E may be --H,--OH, --R, or --OR, and X and Y and Z may be identical or different andselected from --H and --R; R being a C₁ --C₁₆ alkyl, preferably a C₁--C₈ alkyl, which alkyl may be saturated or unsaturated, branched orunbranched and optionally substituted with a carboxy, sulfo or aminogroup; and B and C may be the same or different and selected from C_(m)H_(2m+1;) 1≦m≦5.

BRIEF DESCRIPTION OF THE DRAWING

The present invention is further illustrated by reference to FIG. 1which shows the bleaching of gradually added Acid Blue 45 inphosphate/borate buffer pH 10 at 35° C.; (I): Only dye addition; (II):Dye addition in the presence of Laccase; (III): Dye addition in thepresence of Laccase+Acetosyringone; the experiment conducted asdescribed in Example 8.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of oxidizing a compound with aphenol oxidizing enzyme, characterized by the presence of an enhancingagent of the following formula: ##STR2## in which formula A is a groupsuch as --D, --CH=CH--D, --CH=CH--CH=CH--D, --CH=N--D, --N=N--D, or--N=CH--D, in which D is selected from the group consisting of --CO--E,--SO₂ --E, --N--XY, and --N⁺ --XYZ, in which E may be --H, --OH, --R, or--OR, and X and Y and Z may be identical or different and selected from--H and --R; R being a C₁ -C₁₆ alkyl, preferably a C₁ -C₈ alkyl, whichalkyl may be saturated or unsaturated, branched or unbranched andoptionally substituted with a carboxy, sulfo or amino group; and B and Cmay be the same or different and selected from C_(m) H_(2m+1;) 1≦m≦5.

In a preferred embodiment A in the above mentioned formula is --CO--E,in which E may be --H, --OH, --R, or --OR; R being a C₁ -C₁₆ alkyl,preferably a C₁ -C₈ alkyl, which alkyl may be saturated or unsaturated,branched or unbranched and optionally substituted with a carboxy, sulfoor amino group; and B and C may be the same or different and selectedfrom C_(m) H_(2m+1;) 1≦m≦5.

In the above mentioned formula A may be placed meta to the hydroxy groupinstead of being placed in the paraposition as shown.

In particular embodiments, the enhancing agent is acetosyringone,syringaldehyde, methylsyringate, syringic acid, ethylsyringate,propylsyringate, butylsyringate, hexylsyringate, octylsyringate or ethyl3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate.

The enhancing agent of the invention may be present in concentrations offrom 0.01 to 1000 μM, more preferred 0.1 to 250 μM, most preferred 1 to100 μM.

Preparation of Enhancing Agents

The enhancing agents described in the present application may beprepared using methods well known to those skilled in the art; some ofthe enhancing agents are also commercially available.

We produced methylsyringate, ethylsyringate, propylsyringate,butylsyringate, hexylsyringate and octylsyringate by using the methoddisclosed in Chem. Ber. 67, 1934, p. 67.

Ethyl 3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate was synthesised fromsyringaldehyde and triethyl phosphonoacetate in ethanol/sodiumethanolate. The product was after purification characterised by ¹ H-NMRand ¹³ C-NMR (showing spectra as expected) and the melting point was68-70° C.

Hydrogen peroxide/Oxygen

If the phenol oxidizing enzyme requires a source of hydrogen peroxide,the source may be hydrogen peroxide or a hydrogen peroxide precursor forin situ production of hydrogen peroxide, e.g., percarbonate orperborate, or a hydrogen peroxide generating enzyme system, e.g., anoxidase and a substrate for the oxidase, e.g., an amino acid oxidase anda suitable amino acid, or a peroxycarboxylic acid or a salt thereof.Hydrogen peroxide may be added at the beginning or during the process,e.g., in an amount corresponding to levels of from 0.001-25 mM,particularly to levels of from 0.01-1 mM.

If the phenol oxidizing enzyme requires molecular oxygen, molecularoxygen from the atmosphere will usually be present in sufficientquantity. If more O₂ is needed, additional oxygen may be added.

Phenol Oxidizing Enzyme

In the context of the present invention the enzyme of the phenoloxidizing enzyme may be an enzyme possessing peroxidase activity or alaccase or a laccase related enzyme as described below.

Peroxidases and Compounds possessing Peroxidase Activity

Compounds possessing peroxidase activity may be any peroxidase enzymecomprised by the enzyme classification (EC 1.11.1.7), or any fragmentderived therefrom, exhibiting peroxidase activity, or synthetic orsemisynthetic derivatives thereof (e.g. porphyrin ring systems ormicroperoxidases, cf. e.g. U.S. Pat. No. 4,077,768, EP PatentApplication 537,381, International Patent Applications WO 91/05858 andWO 92/16634).

Preferably, the peroxidase employed in the method of the invention isproducible by plants (e.g. horseradish or soybean peroxidase) ormicroorganisms such as fungi or bacteria. Some preferred fungi includestrains belonging to the subdivision Deuteromycotina, classHyphomycetes, e.g. Fusarium, Humicola, Tricoderma, Myrothecium,Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia,Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672),Humicola insolens, Trichoderma resii, Myrothecium verrucaria (IFO 6113),Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERMP-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli orDreschlera halodes.

Other preferred fungi include strains belonging to the subdivisionBasidiomycotina, class Basidiomycetes, e.g. Coprinus, Phanerochaete,Coriolus or Trametes, in particular Coprinus cinereus f. microsporus(IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g.NA-12) or Trametes (previously called Polyporus), e.g. T. versicolor(e.g. PR4 28-A).

Further preferred fungi include strains belonging to the subdivisionZygomycotina, class Mycoraceae, e.g. Rhizopus or Mucor, in particularMucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales,e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus(IFO 12382) or Streptoverticillum verticillium ssp. verticillium.

Other preferred bacteria include Bacillus humilus (ATCC 12905), Bacillusstearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri,Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonasfluorescens (NRRL B-11).

Further preferred bacteria include strains belonging to Myxococcus, e.g.M. virescens.

The peroxidase may furthermore be one which is producible by a methodcomprising cultivating a host cell transformed with a recombinant DNAvector which carries a DNA sequence encoding said peroxidase as well asDNA sequences encoding functions permitting the expression of the DNAsequence encoding the peroxidase, in a culture medium under conditionspermitting the expression of the peroxidase and recovering theperoxidase from the culture.

Particularly, a recombinantly produced peroxidase is a peroxidasederived from a Coprinus sp., in particular C. macrorhizus or C. cinereusaccording to WO 92/16634.

In the context of this invention, compounds possessing peroxidaseactivity comprise peroxidase enzymes and peroxidase active fragmentsderived from cytochromes, haemoglobin or peroxidase enzymes, andsynthetic or semisynthetic derivatives thereof, e.g., iron porphyrins,and iron phthalocyanines and derivatives thereof.

Determination of Peroxidase Activity (PODU)

1 peroxidase unit (PODU) is the amount of enzyme that catalyzes theconversion of 1 μmole hydrogen peroxide per minute at the followinganalytical conditions: 0.88 mM hydrogen peroxide, 1.67 mM2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate), 0.1 M phosphatebuffer, pH 7.0, incubated at 30° C., photometrically followed at 418 nm.

Laccase and Laccase Related Enzymes

In the context of this invention, laccases and laccase related enzymescomprise any laccase enzyme comprised by the enzyme classification (EC1.10.3.2), any catechol oxidase enzyme comprised by the enzymeclassification (EC 1.10.3.1), any bilirubin oxidase enzyme comprised bythe enzyme classification (EC 1.3.3.5) or any monophenol monooxygenaseenzyme comprised by the enzyme classification (EC 1.14.99.1).

The above mentioned enzymes may be derived from plants, bacteria orfungi (including filamentous fungi and yeasts) and suitable examplesinclude a laccase derivable from a strain of Aspergillus, Neurospora,e.g., N. crassa. Podospora, Botrytis, Collybia, Fomes, Lentinus,Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia,e.g., R. solani, Coprinus, e.g., C. cinereus, C. comatus, C. friesii,and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g.,P. Papilionaceus, Myceliophthora, e.g., M. thermophila, Schytalidium,Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radita (WO 92/01046), orCoriolus, e.g., C. hirsutus (JP 2-238885).

The laccase or the laccase related enzyme may furthermore be one whichis producible by a method comprising cultivating a host cell transformedwith a recombinant DNA vector which carries a DNA sequence encoding saidlaccase as well as DNA sequences encoding functions permitting theexpression of the DNA sequence encoding the laccase, in a culture mediumunder conditions permitting the expression of the laccase enzyme, andrecovering the laccase from the culture.

Determination of Laccase Activity (LACU)

Laccase activity is determined from the oxidation of syringaldazin underaerobic conditions. The violet colour produced is photometered at 530nm. The analytical conditions are 19 μM syringaldazin, 23.2 mM acetatebuffer, pH 5.5, 30° C., 1 min. reaction time.

1 laccase unit (LACU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazin per minute at these conditions.

Industrial Applications

In a preferred embodiment, the method of the is invention findsapplication for bleaching of a textile dye or colorant or textile dyesor colorants in solution.

Colorants and dyes are broad classes of natural and synthetic compounds.The following description and examples of dyes/colorants are notintended to be in any way limiting to the scope of the invention asclaimed:

Synthetic textile dyes bleachable by the method of the invention aretypically azo compounds (with one or several azo, or diazenediyl,groups), as exemplified by Acid Red 151, Direct Blue 1, Direct Brown 44,and Orange II, or anthraquinone compounds, as exemplified by Acid Blue45: ##STR3## Other structural motifs may occur together with these, asexemplified in the formula of Reactive Blue 19: ##STR4##

Some dyes furthermore carry groups capable of coupling to fabricsurfaces (reactive dyes), and some dyes are complexed to metal ions.These modifications will often not influence the applicability of thepresent invention.

A different structure bleachable by the method of the invention is theindigo moiety, here exemplified by the soluble dye indigo carmine:##STR5##

Other dyes and colorants may be of natural origin or may be synthesizedas identical to or resembling natural structures. Examples of categoriesof coloured substances extractable from vegetable sources arepolyphenolic, anthocyanine and carotenoid compounds.

A specific embodiment of the present invention is provided by householdand institutional laundering processes. In such washing and rinsingprocesses, dyes and colorants present on fabrics may leach into thewashing or rinsing liquor and discoloration of the laundry may result.Bleaching of the coloured compounds in solution by the method of theinvention may counteract this undesirable effect. Other systems for dyetransfer inhibition are known in the art (e.g. WO 91/05839).

In another specific embodiment, dyes leached into process water duringtextile processing may be bleached by the method of the invention toprevent undesirable deposition. Other systems are known in the art (e.g.WO 92/18697).

In a third embodiment, the method of the invention finds application inbleaching of pulp for paper production.

Accordingly, the invention provides a method for bleaching oflignin-containing material, in particular bleaching of pulp for paperproduction, which method comprises treatment of the lignin or lignincontaining material with a phenol oxidizing enzyme and an enhancingagent as described in the present invention.

In a fourth embodiment, the method of the invention finds applicationfor lignin modification, e.g., in the manufacture of wood composites,e.g., wood fibre materials such as chipboards, fibre boards, or particleboards, or in the manufacture of laminated wood products, such aslaminated beams and plywood.

In a fifth embodiment, the method of the invention finds application intreatment of waste water, e.g., waste water from the chemical orpharmaceutical industry, from dye manufacturing, from dye-works, fromthe textile industry, or from pulp production (cf. e.g. U.S. Pat. No.4,623,465, or JP-A-2-31887).

In a more specific aspect, the invention provides a method for treatmentof waste water from dye manufacturing, from dye-works, from textileindustry, or from pulp manufacturing, the method comprising treatment ofthe waste water with a phenol oxidizing enzyme in the presence of anenhancing agent of the invention.

In the above mentioned processes and in other applications of theinvention, the enhancing agent may be added at the beginning of theprocess or later, in one or several additions.

According to the invention the phenol oxidizing enzyme may be present inconcentrations of from 0.001-100 mg enzyme protein per liter.

Detergent Compositions

According to the invention, the enhancing agent and the phenol oxidizingenzyme may typically be a component of a detergent composition. As such,it may be included in the detergent composition in the form of adetergent additive. Preferred detergent additive formulations aregranulates, in particular non-dusting granulates, liquids, in particularstabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) and mayoptionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products (polyethyleneglycol,PEG) with mean molecular weights of 1000 to 20000; ethoxylatednonyl-phenols having from 16 to 50 ethylene oxide units; ethoxylatedfatty alcohols in which the alcohol contains from 12 to 20 carbon atomsand in which there are 15 to 80 ethylene oxide units; fatty alcohols;fatty acids; and mono- and di- and triglycerides of fatty acids.Examples of film-forming coating materials suitable for application byfluid bed techniques are given in patent GB 1483591. Liquid enzymepreparations may, for instance, be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acidaccording to established methods. Other enzyme stabilizers are wellknown in the art. Protected enzymes may be prepared according to themethod disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g. as powder, granules, paste or liquid. A liquid detergent maybe aqueous, typically containing up to 70% water and 0-30% organicsolvent, or nonaqueous.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0-50% of anionic surfactant such as linearalkylbenzene-sulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES),secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% ofnonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

The detergent composition may additionally comprise one or more otherenzymes, such as amylases, lipases, cutinases, proteases, andcellulases.

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may additionally contain other bleaching systems which maycomprise a H₂ O₂ source such as perborate or percarbonate which may becombined with a peracid-forming bleach activator such astetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS).Alternatively, the bleaching system may comprise peroxyacids of, e.g.,the amide, imide, or sulfone type.

The enzymes of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative such as, e.g., an aromatic borateester, and the composition may be formulated as described in, e.g., Wo92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents,anti-soil-redeposition agents, dyes, bactericides, optical brighteners,or perfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g., in the range of 7-11.

Particular forms of detergent compositions within the scope of theinvention include:

    ______________________________________    1) A detergent composition formulated as a graulate having    a bulk density of at least 600 g/l comprising    ______________________________________    Linear alkylbenzenesulfonate (cal-                         7-12%    culated as acid)    Alcohol ethoxysulfate (e.g. C.sub.12-18                        1-4%    alcohol, 1-2 EO) or alkyl sulfate    (e.g. C.sub.16-18)    Alcohol ethoxylate (e.g. C.sub.14-15 alco-                        5-9%    hol,    7 EO)    Sodium carbonate (as Na.sub.2 CO.sub.3)                        14-20%    Soluble silicate (as Na.sub.2 O, 2SiO.sub.2)                        2-6%    Zeolite (as NaA1SiO.sub.4)                        15-22%    Sodium sulfate (as Na.sub.2 SO.sub.4)                        0-6%    Sodium citrate/citric acid                         0-15%    (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.7 H.sub.8 O.sub.7)    Sodium perborate (as NaBO.sub.3.H.sub.2 O)                        11-18%    TAED                2-6%    Carboxymethylcellulose                        0-2%    Polymers (e.g. maleic/acrylic acid                        0-3%    copolymer, PVP, PEG)    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. suds                        0-5%    suppressors, perfume, optical    brightener, photobleach)    ______________________________________

    ______________________________________    2) A detergent composition formulated as a granulate having    a bulk density of at least 600 g/l comprising    ______________________________________    Linear alkylbenzenesulfonate (cal-                         6-11%    culated as acid)    Alcohol ethoxysulfate (e.g. C.sub.12-18                        1-3%    alcohol, 1-2 EO) or alkyl sulfate    (e.g. C.sub.16-18)    Alcohol ethoxylate (e.g. C.sub.14-15 alco-                        5-9%    hol, 7 EO)    Sodium carbonate (as Na.sub.2 CO.sub.3)                        15-21%    Soluble silicate (as Na.sub.2 O, 2SiO.sub.2)                        1-4%    Zeolite (as NaA1SiO.sub.4)                        24-34%    Sodium sulfate (as Na.sub.2 SO.sub.4)                         4-10%    Sodium citrate/citric acid                         0-15%    (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.7 H.sub.8 O.sub.7)    Carboxymethylcellulose                        0-2%    Polymers (e.g. maleic/acrylic acid                        1-6%    copolymer, PVP, PEG)    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. suds                        0-5    suppressors, perfume,)    ______________________________________

    ______________________________________    3) A detergent composition formulated as a granulate having    a bulk density of at least 600 g/l comprising    ______________________________________    Linear alkylbenzenesulfonate (cal-                        5-9%    culated as acid)    Alcohol ethoxylate (e.g. C.sub.12-15 alco-                         7-14%    hol, 7 EO)    Soap as fatty acid (e.g. C.sub.16-22 fatty                        1-3%    acid)    Sodium carbonate (as Na.sub.2 CO.sub.3)                        10-17%    Soluble silicate (as Na.sub.2 O, 2SiO.sub.2)                        3-9%    Zeolite (as NaAlSiO.sub.4)                        23-33%    Sodium sulfate (as Na.sub.2 SO4)                        0-4%    Sodium perborate (as NaBO.sub.3.H.sub.2 O)                         8-16%    TAED                2-8%    Phosphonate (e.g. EDTMPA)                        0-1%    Carboxymethylcellulose                        0-2%    Polymers (e.g. maleic/acrylic acid                        0-3%    copolymer, PVP, PEG)    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. suds                        0-5%    suppressors, perfume, optical    brightener)    ______________________________________

    ______________________________________    4) A detergent composition formulated as a granulate having    a bulk density of at least 600 g/l comprising    ______________________________________    Linear alkylbenzenesulfonate (cal-                         8-12%    culated as acid)    Alcohol ethoxylate (e.g. C.sub.12-15 alco-                        10-25%    hol, 7 EO)    Sodium carbonate (as Na.sub.2 CO.sub.3)                        14-22%    Soluble silicate (as Na.sub.2 O, 2SiO.sub.2)                        1-5%    Zeolite (as NaAlSiO.sub.4)                        25-35%    Sodium sulfate (as Na.sub.2 SO.sub.4)                         0-10%    Carboxymethylcellulose                        0-2%    Polymers (e.g. maleic/acrylic acid                        1-3%    copolymer, PVP, PEG)    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. suds                        0-5%    suppressors, perfume)    ______________________________________

    ______________________________________    5) An aqueous liquid detergent composition comprising    ______________________________________    Linear alkylbenzenesulfonate (cal-                        15-21%    culated as acid)    Alcohol ethoxylate (e.g. C.sub.12-15 alco-                        12-18%    hol, 7 EO or C.sub.12-15 alcohol, 5 EO)    Soap as fatty acid (e.g. oleic                         3-13%    acid)    Alkenylsuccinic acid (C.sub.12-14)                         0-13%    Aminoethanol         8-18%    Citric acid         2-8%    Phosphonate         0-3%    Polymers (e.g. PVP, PEG)                        0-3%    Borate (as B.sub.4 O.sub.7)                        0-2%    Ethanol             0-3%    Propylene glycol     8-14%    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g.                        0-5%    dispersants, suds suppressors, per-    fume, optical brightener)    ______________________________________

    ______________________________________    6) An aqueous structured liguid detergent composition    comprising    ______________________________________    Linear alkylbenzenesulfonate                        15-21%    (calculated as acid)    Alcohol ethoxylate (e.g. C.sub.12-15                        3-9%    alcohol, 7 EO, or    C.sub.12-15 alcohol, 5 EO)    Soap as fatty acid (e.g. oleic                         3-10%    acid)    Zeolite (as NaAlSiO.sub.4)                        14-22%    Potassium citrate    9-18%    Borate (as B.sub.4 O.sub.7)                        0-2%    Carboxymethylcellulose                        0-2%    Polymers (e.g. PEG, PVP)    Anchoring polymers such as, e.g.,                        0-3%    lauryl methacrylate/acrylic acid    copolymer; molar ratio 25:1; MW    3800    Glycerol            0-5%    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g.                        0-5%    dispersants, suds suppressors,    perfume, optical brighteners)    ______________________________________

    ______________________________________    7) A detergent composition formulated as a granulate having a    bulk density of at least 600 g/l comprising    ______________________________________    Fatty alcohol sulfate                         5-10%    Ethoxylated fatty acid monoethanol-                        3-9%    amide    Soap as fatty acid  0-3%    Sodium carbonate (as Na.sub.2 CO.sub.3)                         5-10%    Soluble silicate (as Na.sub.2 O, 2SiO.sub.2)                        1-4%    Zeolite (as NaAlSiO.sub.4)                        20-40%    Sodium sulfate (as Na.sub.2 SO.sub.4)                        2-8%    Sodium perborate (as NaBO.sub.3.H.sub.2 O)                        12-18%    TAED                2-7%    Polymers (e.g. maleic/acrylic acid                        1-5%    copolymer, PEG)    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. optical                        0.5%    brightener, suds suppressors, per-    fume)    ______________________________________

    ______________________________________    8) A detergent composition formulated as a granulate    comprising    ______________________________________    Linear alkylbenzensulfonate                         8-14%    (calculated as acid)    Ethoxylated fatty acid monoethanol-                         5-11%    amide    Soap as fatty acid  0-3%    Sodium carbonate (as Na.sub.3 CO.sub.3)                         4-10%    Soluble silicate (as Na.sub.2 O, 2SiO.sub.2)                        1-4%    Zeolite (as NaAlSiO.sub.4)                        30-50%    Sodium sulfate (as Na.sub.2 SO.sub.4)                         3-11%    Sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)                         5-12%    Polymers (e.g. PVP, maleic/acrylic                        1-5%    acid copolymer, PEG)    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. suds                        0-5%    suppressors, perfume)    ______________________________________

    ______________________________________    9) A detergent composition formulated as a granulate    comprising    ______________________________________    Linear alkylbenzenesulfonate                         6-12%    (calculated as acid)    Nonionic surfactant 1-4%    Soap as fatty acid  2-6%    Sodium carbonate (as Na.sub.2 CO.sub.3)                        14-22%    Zeolite (as NaAlSiO.sub.4)                        18-32%    Sodium sulfate (as Na.sub.2 SO.sub.4)                         5-20%    Sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)                        3-8%    Sodium perborate (as NaBO.sub.3.H.sub.2 O)                        4-9%    Bleach activator (e.g. NOBS or                        1-5%    TAED)    Carboxymethylcellulose                        0-2%    Polymers (e.g. polycarboxylate or                        1-5%    PEG)    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. optical                        0-5%    brightener, perfume)    ______________________________________

    ______________________________________    10) An aqueous liquid detergent composition comprising    ______________________________________    Linear alkylbenzenesulfonate                        15-23%    (calculated as acid)    Alcohol ethoxysulfate (e.g. C.sub.12-15                         8-15%    alcohol, 2-3 EO)    Alcohol ethoxylate (e.g. C.sub.12-15 al-                        3-9%    cohol, 7 EO, or    C.sub.12-15 alcohol, 5 EO)    Soap as fatty acid (e.g. lauric                        0-3%    acid)    Aminoethanol        1-5%    Sodium citrate       5-10%    Hydrotrope (e.g. sodium                        2-6%    toluensulfonate)    Borate (as B.sub.4 O.sub.7)                        0-2%    Carboxymethylcellulose                        0-1%    Ethanol             1-3%    Propylene glycol    2-5%    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. polymers,                        0-5%    dispersants, perfume, optical    brighteners)    ______________________________________

    ______________________________________    11) An aqueous liquid detergent composition comprising    ______________________________________    Linear alkylbenzenesulfonate                        20-32%    (calculated as acid)    Alcohol ethoxylate (e.g. C.sub.12-15 alco-                         6-12%    hol,    7 EO, or C.sub.12-15 alcohol, 5 EO)    Aminoethanol        2-6%    Citric acid          8-14%    Borate (as B.sub.4 O.sub.7)                        1-3%    Polymer (e.g. maleic/acrylic acid                        0-3%    copolymer, anchoring polymer such    as, e.g., lauryl    methacrylate/acrylic acid    copolymer)    Glycerol            3-8%    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. hydro-                        0-5%    tropes, dispersants, perfume,    optical brighteners)    ______________________________________

    ______________________________________    12) A detergent composition formulated as a granulate having    a bulk density of at least 600 g/l comprising    ______________________________________    Anionic surfactant (linear                        25-40%    alkylbenzenesulfonate, alkyl sul-    fate, alpha-olefinsulfonate, alpha-    sulfo fatty acid methyl esters,    alkanesulfonates, soap)    Nonionic surfactant (e.g. alcohol                         1-10%    ethoxylate)    Sodium carbonate (as Na.sub.2 CO.sub.3)                         8-25%    Soluble silicates (as Na.sub.2 O, 2SiO.sub.2)                         5-15%    Sodium sulfate (as Na.sub.2 SO.sub.4)                        0-5%    Zeolite (as NaAlSiO.sub.4)                        15-28%    Sodium perborate (as NaBO.sub.3.4H.sub.2 O)                         0-20%    Bleach activator (TAED or NOBS)                        0-5%    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. perfume,                        0-3%    optical brighteners)    ______________________________________

13) Detergent formulations as described in 1)-12 wherein all or part ofthe linear alkylbenzenesulfonate is replaced by (C₁₂ -C₁₈) alkylsulfate.

    ______________________________________    14) A detergent composition formulated as a granulate having    a bulk density of at least 600 g/l comprising    ______________________________________    (C.sub.12 -C.sub.18) alkyl sulfate                         9-15%    Alcohol ethoxylate  3-6%    Polyhydroxy alkyl fatty acid amide                        1-5%    Zeolite (as NaAlSiO.sub.4)                        10-20%    Layered disilicate (e.g. SK56 from                        10-20%    Hoechst)    Sodium carbonate (as Na.sub.2 CO.sub.3)                         3-12%    Soluble silicate (as Na.sub.2 O, 2SiO.sub.2)                        0-6%    Sodium citrate      4-8%    Sodium percarbonate 13-22%    TAED                3-8%    Polymers (e.g. polycarboxylates and                        0-5%    PVP=    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. optical                        0-5%    brightener, photo bleach, perfume,    suds suppressors)    ______________________________________

    ______________________________________    15) A detergent composition formulated as a granulate having    a bulk density of at least 600 g/l comprising    ______________________________________    (C.sub.12 -C.sub.18) alkyl sulfate                        4-8%    Alcohol ethoxylate  11-15%    Soap                1-4%    Zeolite MAP or zeolite A                        35-45%    Sodium carbonate (as Na.sub.2 CO.sub.3)                        2-8%    Soluble silicate (as Na.sub.2 O, 2SiO.sub.2)                        0-4%    Sodium percarbonate 13-22%    TAED                1-8%    Carboxymethyl cellulose                        0-3%    Polymers (e.g. polycarboxylates and                        0-3%    PVP)    Enzymes (calculated as pure enzyme                        0.0001-0.1%    protein)    Minor ingredients (e.g. optical                        0-3%    brightener, phosphonate, perfume)    ______________________________________

16) Detergent formulations as described in 1)-15) which contain astabilized or encapsulated peracid, either as an additional component oras a substitute for already specified bleach systems.

17) Detergent compositions as described in 1), 3), 7), 9) and 12)wherein perborate is replaced by percarbonate.

18) Detergent compositions as described in 1), 3), 7), 9), 12), 14) and15) which additionally contain a manganese catalyst. The manganesecatalyst may, e.g., be one of the compounds described in "Efficientmanganese catalysts for low-temperature bleaching", Nature 369, 1994,pp. 637-639.

19) Detergent composition formulated as a nonaqueous detergent liquidcomprising a liquid nonionic surfactant such as, e.g., linearalkoxylated primary alcohol, a builder system (e.g. phosphate), enzymeand alkali. The detergent may also comprise anionic surfactant and/or ableach system.

The following examples further illustrate the present invention, andthey are not intended to be in any way limiting to the scope of theinvention as claimed.

EXAMPLE 1 Bleaching of Direct Blue 1 with soybean peroxidase with andwithout acetosyringone

A crude soy bean peroxidase (SBP), obtained from Mead Corp., Dayton,Ohio, was purified by anion and cation chromatography followed bygelfiltration to a single protein on SDS-PAGE with an R_(z) -value(A_(404nm) /A_(280nm)) of 2.2:

125 ml of crude SBP were adjusted to pH 7, diluted to 2.3 mS andfiltered through 0.8 μfilter. The sample was applied to 300 ml DEAEcolumn equilibrated with 20 mM phosphate pH 7.0 and the peroxidaseeluted with a 1 M NaCl linear gradient in the same buffer. Fractionswith peroxidase activity were pooled.

Pooled fractions from anion exchange chromatography (190 ml) wereconcentrated and washed by ultrafiltration (GR61PP membrane from Dow,Denmark). pH was adjusted to 5.3 ionic strength to 2.3 mS in the samplebefore application to a 200 ml S-Sepharose column previouslyequilibrated with 50 mM acetate pH 5.3. The effluent containing theperoxidase activity was concentrated and washed by ultrafiltration to afinal volume of approx. 10 ml.

A 5 ml concentrated sample from cation exchange chromatography wasapplied to a 90 cm Sephacryl S-200 column equilibrated and eluted with0.1 M acetate pH 6.1. Fractions with peroxidase activity giving only oneband on SDS-PAGE were pooled.

The bleaching rate of Direct Blue 1 (DB1) by the purified SBP wasdetermined using an enhancer according to the invention. The followingconditions were used:

    ______________________________________                         Final concentration    ______________________________________    200 μl 50 mM Britton-Robinson buffer*                           10 mM    pH 6, 8 and 10, respectively    200 μl DB1 ˜ 3.0 Abs. Units (610 nm)                           0.6 (A.sub.610nm)    200 μl SBP with A.sub.404nm = 0.0005 at pH 6                           0.0001 or    and 8 or with A.sub.404nm = 0.005 at pH 10                           0.001 (A.sub.404nm)**    200 μl 50 μM enhancer                           10 μM    200 μl 100 μM H.sub.2 O.sub.2                           20 μM    ______________________________________     *(50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted     to the value of interest with NaOH).     **corresponding to approximately to 0.04 mg/l and 0.4 mg/l.

Reagents were mixed in a thermostated cuvette at 30° C. and thebleaching was started by addition of hydrogen peroxide. The bleachingwas detected spectrophotometrically at 610 nm, which is the wavelengthof the absorption peak of DB1. Bleaching was followed for 4 minutes, andthe reduction in absorbance (100x(A_(610nm),start-A_(610nm),4min.)/A_(610nm),start %) was determined.

A_(610nm),start was determined by replacement of hydrogen peroxide withwater.

                  TABLE 1    ______________________________________    Bleaching of Direct Blue 1 with SBP in 4 Minutes              % DB1 bleaching in 4 min.                                   pH 10    Enhancer    pH 6        pH 8   10x SBP!    ______________________________________    No          0.7         <0.7   <0.7    acetosyringone                19.8        20.0   3.3    ______________________________________

From the results presented in Table 1 above, it appears that by addingan enhancer of the invention a much faster bleaching of the dye isobtained compared to the experiment without enhancer.

EXAMPLE 2 Bleaching of Direct Blue 1 with Coprinus cinereus peroxidaseand without enhancers

A Coprinus cinereus peroxidase (CiP), obtained as described in WO9412621 was used.

Dilutions of CiP were made in a solution of 0.15 gram/l of Triton X-405.

The bleaching rate of Direct Blue 1 (DB1) by purified CiP was determinedusing the following conditions:

    ______________________________________                        Final concentration    ______________________________________    200 μl 50 mM Britton-Robinson buffer*                          10 mM    200 μl DB1 ˜ 3.0 Abs. Units (610 nm)                          0.6 (A.sub.610nm)    200 μl 0.40 mg/l CiP (pH 8.5)                          0.08 mg/l (pH 8.5) or    0.80 mg/l CiP (pH 10.5)                          0.16 mg/l (pH 10.5)    200 μl 25 μM enhancer                          5 μM    200 μl 100 μM H.sub.2 O.sub.2                          20 μM    ______________________________________     *(50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted     to the value of interest with NaOH).

Reagents were mixed in a thermostated cuvette at 30° C. and thebleaching was started by addition of hydrogen peroxide. The bleachingwas detected spectrophotometrically at 610 nm, which is the wavelengthof the absorption peak of DB1. Bleaching was followed for 1 minute, andthe initial reduction in absorbance, --ΔmAbs/minute, was determined.

                  TABLE 2    ______________________________________    Initial Bleaching of Direct Blue 1 with CiP    Enhancer      -ΔmAbs/minute    pH:              8.5     10.5    ______________________________________    Acetosyringone   239     1    Syringaldehyde   151     4    Methylsyringate  245     8    No enhancer      2       0    ______________________________________

From the results presented in Table 2 above it appears that by adding anenhancer of the invention a much faster bleaching of the dye compared tothe experiment without enhancer is obtained. Even at pH 10.5 asignificant bleaching with an enhancer of the invention is obtained,whereas no bleaching at all can be seen without the addition of anenhancer.

EXAMPLE 3 Bleaching of Chicago Sky Blue 6B (CSB) with Coprinus cinereusperoxidase and enhancers

Bleaching tests were performed in exactly the same way as described inExample 2 except that instead of using DB1 Chigaco Sky Blue (CSB)(obtainable from Aldrich) was used, and the following enhancers weretested:

methylsyringate

ethylsyringate

propylsyringate

butylsyringate

hexylsyringate

octylsyringate

ethyl 3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate.

The following results were obtained:

                  TABLE 3    ______________________________________    Initial Bleaching of CSB with CiP    Enhancer        -ΔmAbs/minute    pH:                8.5     10.5    ______________________________________    methylsyringate    211     42    ethylsyringate     240     52    propylsyringate    228     60    butylsyringate     228     48    hexylsyringate     276     36    octylsyringate     192     15    ethyl 3-(4-hydroxy-3,5-                       48      48    dimethoxyphenyl)acrylate    No enhancer        8       6    ______________________________________

EXAMPLE 4

Bleaching of Direct Blue 1 (DB1) using various Coprinaceae laccases andmethylsyringate at pH 5.5-8.5

Bleaching of the dye Direct Blue 1 at various pH values was conductedusing a laccase obtained from Coprinus comatus, Coprinus friesii,Coprinus plicatilis, Panaeolus papilionaceus or Psathyrella condolleanaand methylsyringate.

The above mentioned strains were fermented in the following way:

The strains were inoculated on PDA agar plates (PDA: 39 g/l potatodextrose agar) and grown at 26° C. for 3 days. Shake flasks were theninoculated with 6-8 small squares (⁻ 0.5 cm×0.5 cm) of agar containingmycelium and fermented for 3-10 days at 26° C. and 200 rpm using thefollowing medium:

    ______________________________________                Deposit no.                         Medium  Growth    ______________________________________    Coprinus comatus*                  CBS 631.95 A       10 days    Coprinus friesii                  CBS 629.95 A       3 days    Panaeolus     CBS 630.95 A       10 days    papilionaceus    Psathyrella   CBS 628.95 B       7 days    condolleana    Coprinus plicatilis                  CBS 627.95 A       8 days    ______________________________________     *All the strains mentioned in this Example have been deposited according     to the Budapest Treaty on the International Recognition of the Deposits o     Microorganisms for the Purpose of Patent Procedures, on 16 August 1995, a     Centraalbureau voor Schimmelcultures, Oosterstraat 1, Postbus 273, NL3740     AG Baarn, Netherlands, under the above mentioned Accession numbers.

    ______________________________________    Media:    ______________________________________    A:          soja meal       30       g/l                maltodextrin    15       g/l                bacto peptone   5        g/l                pluronic        0.2      g/l    B:          potato meal     50       g/l                barley meal     25       g/l                BAN 800MG*      0.025    g/l                Na-caseinate    5        g/l                crushed soja    10       g/l                Na2HPO4, 12 H2O 4.5      g/l                Pluronic        0.05     ml/l    ______________________________________     *BAN 800MG obtainable from Novo Nordisk A/S.

After fermentation the culture broths were centrifugated and thesupernatants were used in the tests described below.

The bleaching rate of DB1 was determined using the following conditions:

    ______________________________________                         Final concentration    ______________________________________    400 μl 50 mM Britton-Robinson buffer*,                           20 mM    (pH 5.5, 7.0, and 8.5 respectively),    200 μl DB1 ˜ 3.0 Abs. Units (610 nm)                           0.6 (A.sub.610nm)    200 μl 50 μM methylsyringate                           10 μM    200 μl laccase               at pH 5 and 7:  4 LACU/l               at pH 8.5:      20 LACU/l    ______________________________________     *(50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted     to the value of interest with NaOH).

Reagents were mixed in a 1 ml thermostated cuvette at 30° C. and thebleaching was started by addition of the laccase.

The bleaching was followed spectrophotometrically at 610 nm, which isthe wavelength of the absorption peak of DB1, with readings every 5 sec.for a period of 5 minutes. The initial bleaching rate was determinedfrom the first linear part of the absorbance curve.

The following results were obtained with methylsyringate:

    ______________________________________    Laccase:      -ΔmAbs/minute    pH:           5.5         7.0   8.5    ______________________________________    C. comatus    33          23    2    C. friesii    40          55    61    Pan. papilionaceus                  16          19    18    Ps. condolleana                  45          54    43    C. plicalitis 42          39    14    ______________________________________

The following results were obtained with no enhancer:

    ______________________________________    Laccase:     -ΔmAbs/minute    pH:          5.5          7.0   8.5    ______________________________________    C. comatus   0            0     0    C. friesii   0            0     0    Ps. condolleana                 0            0     0    C. plicalitis                 0            0     0    ______________________________________

EXAMPLE 5 Bleaching of Direct Blue 1 (DB1) using Coprinus cinereuslaccase with/without enhancing agents at pH 5.5-8.5

Bleaching of the dye Direct Blue 1 at various pH values was conductedusing Coprinus cinereus laccase and one of the following enhancingagents:

None

acetosyringone

syringaldehyde

methylsyringate.

The laccase was obtained in the following way: Coprinus cinereus (IFO30116--freely available to the public from Institute of Fermentation,Osaka (IFO) under the indicated deposit number) was inoculated from aPDA agar slant (PDA: 39 g/l potato dextrose agar) into a 100 ml shakeflask containing medium A (Medium A is described in Example 3). Theculture was cultivated for 6 days at 26° C. and 100 rpm. A 10-literfermentor containing medium A was inoculated with the 100 ml culturebroth. The fermentation ran for 6 days at 26° C. and 100 rpm. Theculture broth was filtrated and concentrated by ultrafiltration. Furtherpurification was carried out using hydrophobic interactionchromatography followed by anionic exchange chromatography. This processresultated in a preparation with a laccase activity of 3.6 LACU/ml. Theestimated purity was >80% on a protein basis.

The bleaching rate of DB1 was determined using the owing conditions:

    ______________________________________                         Final concentration    ______________________________________    400 μl 50 mM Britton-Robinson buffer*,                           20 mM    (pH 5.5, 7.0 and 8.5 respectively),    200 μl DB1 ˜ 3.0 Abs. Units (610 nm)                           0.6 (A.sub.610nm)    200 μl 50 μM enhancing agent                           10 μM    200 μl C. cinereus laccase                           1 mg/l    ______________________________________     *(50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted     to the value of interest with NaOH).

Reagents were mixed in a 1 ml thermostated cuvette at 30° C. and thebleaching was started by addition of the laccase.

The bleaching was followed spectrophotometrically at 610 nm, which isthe wavelength of the absorption peak of DB1 with readings every 5 sec.for a period of 5 minutes. The initial bleaching rate was determinedfrom the first linear part of the absorbance curve.

The following results were obtained:

    ______________________________________    Enhancing agent                 -ΔmAbs/minute    pH:          5.5          7.0   8.5    ______________________________________    none         13           5     3    aceto-       28           94    50    syringone    syring-      29           79    28    aldehyde    methyl-      20           94    57    syringate    ______________________________________

EXAMPLE 6 Bleaching of Direct Blue 1 (DB1) using Coprinus cinereuslaccase and acetosyringone

Bleaching of the dye Direct Blue 1 at various pH values was conductedusing Coprinus cinereus laccase and the enhancing agent acetosyringone.

The laccase was obtained as described in Example 5.

The bleaching rate of DB1 was determined using the following conditions:

    ______________________________________                         Final concentration    ______________________________________    400 μl 50 mM Britton-Robinson buffer*,                           20 mM    (pH 4, 5, 6, 7, and 8 respectively),    200 μl DB1 ˜ 3.0 Abs. Units (610 nm)                           0.6 (A.sub.610nm)    200 μl 50 μM acetosyringone                           10 μM    200 μl C. cinereus laccase                           3.2 mg/l    ______________________________________     *(50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted     to the value of interest with NaOH).

Reagents were mixed in a 1 cm thermostated cuvette at 30° C. and thebleaching was started by addition of the laccase.

The bleaching was detected spectrophotometrically at 610 nm, which isthe wavelength of the absorption peak of DB1. After 5 sec. bleaching wasfollowed for 4 minutes.

The following results were obtained:

    ______________________________________               Initial DB1 bleaching               (-ΔmAbs/ min)           pH  (% of pH 7-value)    ______________________________________           4   18%           5   13%           6   35%           7   100%           8   69%    ______________________________________

It can be seen from the results given above that the optimum bleachingis achieved at pH around 7, but the system also shows an effectivebleaching at pH 8.

EXAMPLE 7 Bleaching of Direct Blue 1 with Trametes villosa laccase withand without enhancing agents

Laccase obtained from Trametes villosa: 800 ml culture broth of Trametesvillosa, CBS 678.70, was filtered with filter aid to give a clearfiltrate, which was concentrated and washed by ultrafiltration on amembrane with a cut-off of 6-8 kDa. One ml samples of concentratedpreparation was applied onto a Q-Sepharose HP column (Pharmacia, Sweden)equilibrated with 0.1 M fosfate pH 7, and the laccase was eluted with aflat NaCl gradient around 0.25 M. Fractions with laccase activity from10 runs were pooled and concentrated by ultrafiltration to an activityof 500 LACU/ml.

The following conditions were used:

    ______________________________________                         Final concentration    ______________________________________    400 μl 50 mM Britton-Robinson buffer*,                           20 mM    pH 5.5 and pH 7.0 respectively,    200 μl DB1 ˜ 3.0 Abs. Units (610 nm)                           0.6 (A.sub.610nm)    200 μl 50 μM enhancer                           10 μM    200 μl Enzyme dilution    ______________________________________     *(50 nM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted     to the value of interest with NaOH).

Reagents were mixed in a 1 cm thermostated cuvette at 30° C. and thebleaching was started by addition of enzyme.

The bleaching was detected spectrophotometrically at 610 nm, which isthe absorption peak of DB1. After 5 sec. bleaching was followed for 4minutes.

From the results presented below, it appears that adding enhancers ofthe invention a much faster bleaching of the dye can be obtainedcompared to the experiment without enhancer. Enzyme dosages given are inthe final incubation mixture.

Bleaching of Direct Blue 1 with Trametes villosa laccase, obtained asdescribed above, at pH 5.5 (1.6 mg/l) and pH 7.0 (16 mg/l):

    ______________________________________              DB1 bleaching in 4 minutes              (-ΔmAbs/4 min)    Enhancer       pH 5.5    pH 7.0    ______________________________________    No enhancer    0         0    Acetosyringone 447       242    Syringaldehyde 438       112    ______________________________________

EXAMPLE 8 Bleaching of gradually added Acid Blue 45 with Coprinuscinereus laccase with and without enhancing agent

Ideally, dye transfer inhibition systems for laundry applications shouldbe tested in a real wash where dyed fabrics give off dyes to the washsolution as a result of the combined action of the detergent,temperature and mechanical agitation taking place.

To simulate such a process, however, a magnetically stirred beaker wasused as the reaction vessel and dye was added gradually from a stocksolution (using a Metrohm 725 dosimat). The solution was monitoredspectrophotometrically using a Zeiss multichannel spectrometer (MCS)equipped with a fibre-optics immersion probe.

Stock solutions of acetosyringone was prepared in a suitablewater/ethanol mixture. Stock solutions of the anthraquinone dye AcidBlue 45 were made with water.

The laccase was recovered from a 10-liter fermentation of Coprinuscinereus (IFO 30116) as described in Example 4.

The following conditions were used in the experiment:

Temperature: 35° C.

Medium and pH: 50 nM/50 mM phosphate/borate buffer at pH 10

Acetosyringone (when applicable): 10 μM

Laccase: 10 mg/l

Dye addition program: linear addition at a rate of ca 0.34 bs/40 min,referring to the absorbance of Acid Blue 45 at its maximum absorbancewavelength (590 nm for Acid Blue 45).

FIG. 1 shows the results of the bleaching tests. The following symbolsare used: (I): Only dye addition; (II): Dye addition in the presence ofLaccase; (III): Dye addition in the presence of Laccase+acetosyringone.

It can be seen from FIG. 1 that the bleaching effect is enhanced byacetosyringone.

EXAMPLE 9 Dye Transfer Inhibition Using Coprinus cinereus Laccase

A small-scale experiment was carried out in which clean cotton testpieces were washed together with dyed fabrics bleeding dye into the washsolution, the experiment conducted in the absence and in the presence oflaccase and enhancing agent.

After wash, the Hunter colour difference between the above mentionedcotton pieces and clean cotton pieces (washed in the absence of bleedingfabrics) was measured and taken as a measure of the degree of dyetransfer resulting from the wash.

Materials used:

Bleeding fabrics dyed with Acid Red 151 (AR 151) or Direct Blue 1 (DB1).

Clean white cotton (bleached, no optical brightener added).

Liquid detergent and powder detergent as typically met in the NorthAmerican market place; both detergents contained no bleaching system.

Coprinus cinereus laccase, obtained as described in Example 4.

Washing procedure:

The washing processes were carried out in beakers with magneticalstirring at 35° C. for 15 min., after which the test fabrics were rinsedthoroughly in tap water and air-dried overnight in the dark before theHunter readings were taken by using a Datacolor Elrephometer 2000reflectance spectrometer.

Laccase system: Laccase at a level of 10 mg/l with the enhancing agentacetosyringone at a level of 10 μM.

The following results were obtained:

Wash in liquid detergent solution (2 g/l, water hardness 6° dH) at pH8.5:

    ______________________________________                Hunter colour difference (delta E)                with respect to white, washed cotton                 Cotton washed                           Cotton washed                 with AR 151                           with DB 1                 bleeders  bleeders    ______________________________________    Wash with no laccase                   12          26    system    Wash with laccase                   1           7    system    ______________________________________

Wash in powder deterrent solution (1 g/l, water hardness 6° dH) at pH10.0:

    ______________________________________                Hunter colour difference (delta E)                with respect to white, washed cotton                 Cotton washed                           Cotton washed                 with AR 151                           with DB 1                 bleeders  bleeders    ______________________________________    Wash with no laccase                   21          29    system    Wash with laccase                   4           8    system    ______________________________________

Typical significant differences in the delta E readings are 2-3 units,so the data reflect significant reduction of dye transfer with thelaccase treatments relative to the treatment with no laccase system.

EXAMPLE 10 Dye Transfer Inhibition Using Myceliophthora thermophilaLaccase

A small-scale experiment was carried out in which clean cotton testpieces were washed together with dyed fabrics bleeding dye into the washsolution, the experiment conducted in the absence and in the presence oflaccase and enhancing agent.

After wash, the Hunter colour difference between the above mentionedcotton pieces and clean cotton pieces (washed in the absence of bleedingfabrics) was measured and taken as a measure of the degree of dyetransfer resulting from the wash.

Materials used

Bleeding fabrics dyed with Acid Red 151 (AR 151) or Direct Blue 1 (DB1).

Clean white cotton (bleached, no optical brightener added).

Liquid detergent (No. 1) as typically met in the European market place;liquid detergent (No. 2) as typically met in the North American marketplace.

Myceliophthora thermophila laccase, produced as described inPCT/US95/06815).

Washing procedure

The washing processes were carried out in beakers with magneticalstirring at 35° C. for 15 min., after which the test fabrics were rinsedthoroughly in tap water and air-dried overnight in the dark before theHunter readings were taken by using a Datacolor Elrephometer 2000reflectance spectrometer.

Laccase systems: M. thermophila laccase at a level of 0.87 mg/l with theenhancing agent acetosyringone (AS) or the enhancing agentmethylsyringate (MS) at a level of 10 μM.

The following results were obtained:

Wash in solution of liquid detergent No. 1 (7 g/l. water hardness 12°dH) at an initial pH of 7.0:

    ______________________________________                  Hunter colour difference (delta E)                  with respect to white, washed cotton                  Cotton washed                            Cotton washed                  with AR 151                            with DB 1                  bleeders  bleeders    ______________________________________    Wash with no laccase                    7           27    system    Wash with AS-based laccase                    5           13    system    Wash with MS-based laccase                    4           12    system    ______________________________________

Wash in solution of liquid detergent No. 2 (2 g/l, water hardness 6° dH)at pH 8.5:

    ______________________________________                  Hunter colour difference (delta E)                  with respect to white, washed cotton                  Cotton washed                            Cotton washed                  with AR 151                            with DB 1                  bleeders  bleeders    ______________________________________    Wash with no laccase                    14          29    system    Wash with AS-based laccase                    5           10    system    Wash with MS-based laccase                    3           8    system    ______________________________________

Typical significant differences in the delta E readings are 2-3 units,so the data reflect significant reduction of dye transfer with thelaccase treatments relative to the treatment with no laccase system.

We claim:
 1. A method for bleaching dyes or colorants in a solution,comprising treating the dyes or colorants in the solution with a phenoloxidizing enzyme and an enhancing agent of the following formula:##STR6## in which formula A is a group such as --D, --CH=CH--D,--CH=CH--CH=CH--D, --CH=N--D, --N=N--D, or --N=CH--D, in which D isselected from the group consisting of --CO--E, --SO₂ --E, --N--XY, and--N+--XYZ, in which E may be --H, --OH, --R, or --OR, and X and Y and Zmay be identical or different and selected from --H and --R; R being aC₁ -C₁₆ alkyl, which alkyl may be saturated or unsaturated, branched orunbranched and optionally substituted with a carboxy, sulfo or aminogroup; and B and C may be the same or different and selected from C_(m)H_(2m+1;) 1≦m≦5.
 2. A method according to claim 1, in which theenhancing agent is selected from the group consisting of acetosyringone,syringaldehyde, methylsyringate and syringic acid.
 3. A method accordingto claim 1, in which the phenol oxidizing enzyme is a peroxidase and ahydrogen peroxide source.
 4. A method according to claim 3, wherein theperoxidase is horseradish peroxidase, soybean peroxidase or a peroxidaseenzyme derived from Coprinus, Bacillus, or Myxococcus.
 5. A methodaccording to claim 3, wherein the hydrogen peroxide source is hydrogenperoxide or a hydrogen peroxide precursor, or a hydrogen peroxidegenerating enzyme system, or a peroxycarboxylic acid or a salt thereof.6. A method according to claim 1, in which the phenol oxidizing enzymeis a laccase enzyme together with oxygen.
 7. A method according to claim6, wherein the laccase is derived from Trametes, or Coprinus.
 8. Amethod according to claim 1, in which said method is a method forinhibiting the transfer of a textile dye from a dyed fabric to anotherfabric when said fabrics are washed together in a wash liquor.
 9. Amethod according to claim 8, in which the enhancing agent is added atthe beginning of, or during the process.
 10. A method according to claim8, in which the concentration of the enhancing agent is in the range offrom 0.01-1000 μM.